Multiplexed analysis of analytes is an important tool in biomedical discovery such as drug development, genome analysis, and diagnostics. An exemplary use of multiplexed analysis is the study of the human genome structure and expression. Recent study of the human genome has demanded simultaneous study of many genomic sites instead of serially studying individual sites. Particularly important to multiplexed genomic analysis are tools such as nucleic acid arrays commonly known as DNA chips.
Although the basic principles behind microarrays are sound, the manufacture and analysis is expensive and complex. As a result, while the number of potential applications is great, few laboratories can afford the technology for their diagnostic or research goals. The described invention addresses this discrepancy allowing for multiplexed analysis that will not have the cost prohibitions of current microarray products.
Arrays are also used in drug discovery, for example, by identifying gene expression of human cells and their response to drugs, hormones, inhibitors, enzymes, and other molecules. Although the basic principles behind arrays are sound, previously described methods are difficult and costly to manufacture and analysis is often expensive and complex. Signature patterns of expression may indicate new drug targets, permit rapid screening for drugs of desired effect, and potentially reduce the time from bench to bedside. One of the most important applications of microarrays will probably be in the field of pharmacogenetics. Pharmacogenetics is the study of how an individual's genetics can affect the probability of different treatment outcomes and how the response to a medication can differ based on an individual's genetically determined metabolic constitution. These differences arise from polymorphisms (minor differences in gene sequences) in the genes responsible for the actual drug target or in genes that direct metabolic enzymes that activate, deactivate, or alter the drug in the body. Microarrays will be used during the drug discovery process, the screening of participants in clinical drug trials, and very likely as part of the standard clinical work up of patients.
Multiplexed analysis of analyte samples may be achieved by parallel processing. In particular, reactions where an analyte will selectively react with a sub-population compound from a larger population of different compounds, are ideally suited for parallel analysis. For example, U.S. Pat. No. 5,744,305, herein incorporated in its entirety by reference, describes the use of a collection of compounds arrayed on a planar surface, where particular compounds are synthesized at particular regions on the planar surface. The array is then contacted with an analyte such that certain compounds in the analyte will specifically bind an array compound.
Existing array methods require arraying compounds by situating such compounds onto surfaces, for example, a glass slide, in predefined different locations either by spotting preformed compounds, or by synthesizing compounds in-situ. Compound identity is maintained solely by its position upon the array surface. Accordingly, the entire array must remain intact for the duration of the analysis. For example, compound identity would be lost if the array were sectioned into individual compound sections that were then randomly shuffled. It is impossible, therefore, to recreate the original array without knowledge of the chemical identity of each compound section. A shuffled array may be reconstructed, however, if the chemical identity of each compound section could be ascertained. Direct analysis is unlikely since the amount of compound present within a compound section is often too minute. If a unique and detectable code is associated with each compound section, then the code would correlate to a particular compound, or region within the array, from which the compound section was derived. A coded compound section then comprises a substrate linking a compound and a code. Encoding compounds imparts portability upon the compound not found with unencoded compound arrays.
Arrays can be in the form of two-dimensionally distributed microscopic spots of nucleic acid material deposited on a solid matrix, usually a microscopy slide. The task of depositing thousands of these spots requires automation. One approach to automation is to print arrays by using computer controlled high-speed robotics. Here, pre-formed different DNA probe regions are produced by first amplifying target DNA by PCR. Next, minute samples of the now amplified DNA are transferred to glass slides using a robotic printer head. Glass slides are pre-coated with a chemical linker that will retain the probe DNA spots in place despite heat denaturation. Standardization and reproducibility of array spotting is difficult to achieve—by printing arrays because of the source of the molecules and the method for their deposition. For example, DNA can be viscous and therefore hard to deliver accurately through the narrow channels of a typical print head.
When arrays are manufactured with print heads, the print heads must first be filled with different samples of probe DNA, and then the head is moved for deposition on slides. This requires the use of computerized robotics to direct the print head to go back and forth between the source of DNA, particular coordinates on the solid matrix (glass slide), and washing and drying stations. The printing speed allows 20-60 arrays each containing 4000 compound locations to be manufactured in 3-4 hours. Scalability is accomplished by simultaneously printing more arrays. This, of course, necessitates additional expensive spotting systems, thus raising costs.
An alternative to printed arrays is the use of light-directed synthesis to construct high-density DNA probe arrays (or DNA chips). Instead of depositing DNA solutions on a slide surface, the DNA is formed in situ by synthesizing a desired DNA sequence directly onto a solid support. The solid support typically contains a covalent linker molecule with a photolabile-protecting group. By selectively applying light to some sites and not others, the light exposed sites become activated. Activated sites then react with protected nucleotides while the inactivated sites remain unaltered. This cycle can be repeated several times using different masks and thus producing a high-density two-dimensional matrix containing different sequence probes. Complex DNA mixtures are then analyzed by correlating active compounds to their fixed position within the two dimensional array.
DNA applied to array surfaces can be derived from fully or partially sequenced DNA clones, EST's (Expressed Sequence Tags), or any cDNA chosen from a library. A two-color hybridization scheme is typically used to monitor the presence or amplification of the DNA regions of interest. Two-color analysis provides for comparison of two DNA sources. For example, in CGH (Comparative Genomic Hybridization), one source of DNA is the test DNA and the other is the reference DNA. After these two fluorescently labeled sets of DNA are hybridized to the array, the resulting ratio of fluorescence intensities at a given spot can be quantified. This measurement then yields a ratio of copy number corresponding to the reference and test DNA associated with that particular DNA or probe region of the array.
Both spotted and in-situ arrays must be individually manufactured by expensive and often temperamental equipment. In-situ synthesis requires hours of stepwise reactions to create one individual compound array. Even if multiple arrays are synthesized simultaneously, the process is machine and mask limited. Moreover, each time a new array compound pattern is desired, a new set of masks must be fashioned. This problem magnifies as higher densities of different compounds are placed onto the array surface. Since compound identity information is strictly positional, highly accurate placement of individual compounds is an absolute and non-trivial requirement for fabricating high-density arrays.
The invention described herein overcomes these and other problems with present array technology.